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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Publications at this Location » Publication #211425
Title: Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples

Author
item Traul, Donald
item Taus, Naomi
item OAKS, J - WSU
item O'TOOLE, D - UNIVERSITY OF WYOMING
item RURANGIRWA, F - WSU
item BASZLER, T - WSU
item Li, Hong

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2007
Publication Date: 7/1/2007
Citation: Traul, D., Taus, N.S., Oaks, J.L., O'Toole, D., Rurangirwa, F.R., Baszler, T.V., Li, H. 2007. Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples. Journal of Veterinary Diagnostic Investigation. 19:405-408

Interpretive Summary: Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain animals caused by ovine herpesvirus 2 (OvHV-2), a herpesvirus carried by sheep, can pose a significant diagnostic challenge to veterinary clinicians in that its clinical signs is difficult to differentiate from several other common viral diseases. The previous development of a nested PCR assay specific for the virus advanced the ability to detect OvHV-2 DNA in animals with clinical MCF. However, the use of this PCR assay as a routine diagnostic method for clinical MCF in veterinary diagnostic laboratories is problematic because the nested amplification format has significant potential for false-positive due to amplified DNA contamination, although it is highly sensitive. In this report, we validated a non-nested and a real-time PCR for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of tissue or blood samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of the disease; 2) 207 tissue samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples from clinically affected animals, 95 (98%) were positive by non-nested PCR and 93 (96%) were positive by real-time PCR, respectively. All 207 tissue samples from the animals with experimentally induced MCF were positive by real-time PCR. Neither non-nested PCR nor real-time PCR detected any sample from 100 animals without any evidence of clinical MCF. The data confirmed that both non-nested and real-time PCR maintained high specificity and had good sensitivity for the detection of OvHV-2 DNA in clinical samples from animals with SA-MCF.

Technical Abstract: Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants caused by ovine herpesvirus 2 (OvHV-2), can pose a significant diagnostic challenge to veterinary clinicians in that its clinical presentation is difficult to differentiate from several other common viral diseases. The previous development of a nested PCR assay specific for the virus advanced the ability to detect OvHV-2 DNA in animals with clinical MCF. However, the use of this PCR assay as a routine diagnostic method for clinical MCF in veterinary diagnostic laboratories is problematic because the nested amplification format has significant potential for amplicon contamination. In this report, we validated a non-nested and a real-time PCR for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of tissue or blood samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 207 tissue samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by non-nested PCR and 93 (96%) were positive by real-time PCR, respectively. All 207 tissue samples from the animals with experimentally induced MCF were positive by real-time PCR. Neither non-nested PCR nor real-time PCR resulted in a positive signal from any of the 100 samples from animals without any evidence of clinical MCF. The data confirmed that both non-nested and real-time PCR maintained high specificity and had adequate sensitivity for the detection of OvHV-2 DNA in clinical samples from animals with SA-MCF.